ABSTRACT Both red snow crab (Chionoecetes japonicus Rathbun, 1932)
and snow crab (Chionoecetes opilio Fabricius, 1788) are commercially
important species in Korea. The geographical ranges of the two species
overlap in the East Sea, where both species are fished commercially.
Morphological identification of the two species and putative hybrids can
be difficult because of their overlapping morphological characteristics.
The presence of putative hybrids can affect the total allowable catch
(TAC) of C. japonicus and C. opilio, and causes problems managing C.
japonicus and C. opilio wild resources. To date, however, no natural
hybridization has been reported between C. japonicus and C. opilio,
despite their overlapping distributions along the coast of the East Sea.
In this study, the internal transcribed spacer (ITS) region of major
ribosomal RNA genes from the nuclear genome and the cytochrome oxidase I
(CO I) gene from the mitochondrial genome were sequenced to determine
whether natural hybridization occurs between the two species. Our
results revealed that all putative hybrids identified using
morphological traits had two distinct types of ITS sequences
corresponding to those of both parental species. Mitochondrial CO I gene
sequencing showed that all putative hybrids had sequences identical to
C. japonicus. A genotyping assay based on single nucleotide
polymorphisms in the ITS 1 region and the CO I gene produced the most
efficient and accurate identification of all hybrid individuals.
Molecular data clearly demonstrate that natural hybridization does occur
between C. japonicus and C. opilio, but only with C. japonicus as the
maternal parent.

Red snow crab (Chionoecetes japonicus) and snow crab (Chionoecetes
opilio) are the most important commercial species in Korean fisheries
(Chun et al. 2008, National Fisheries Research and Development Institute
2008). Because of its growing economic importance, snow crab harvesting
became widespread, and developed into an intensive commercial fishery
around the late 1990s, reaching a peak production of 4,817 tons in 2007.
This equates to more than 47 billion won (US$43 million) (KOSTAT 2009).
The average annual production of red snow crab between 2007 and 2009 was
27,891 tons, which corresponds to more than 45 billion won (US$41
million).

In the East Sea, where Chionoecetes species occur, snow crabs are
common at depths ranging from 200 500 m, whereas red snow crabs are
found at depths from 400 2,000 m (Kort 1980, Yamasaki & Kuwahara
1991, National Fisheries Research and Development Institute 2008). The
geographical ranges of snow crabs and red snow crabs overlap in the East
Sea in areas where commercial fisheries are focused. The spawning season
of snow crabs extends from March to April, and that of red snow crabs
extends throughout the year (Yosho & Hayashi 1994, Lim et al. 2000,
Chun et al. 2008). The two species are very similar morphologically, but
in general can be distinguished by their carapace and dorsal coloration.
The fresh color of snow crab is dark brown whereas that of red snow crab
is uniformly vermilion. There are also some differences in relative
carapace strength/solidity between the species, with snow crab carapaces
being softer (National Fisheries Research and Development Institute
2008).

For fishery management purposes, commercial snow crab and red snow
crab fisheries target only the largest males. For snow crabs,
regulations impose a minimum carapace width of 90 mm for males, and
prohibit female landings. Moreover, fisheries of these species in Korea
are managed under a total allowable catch (TAC) system and include
no-take periods of June through November for snow crabs and July 10
through August 20 for red snow crabs. The National Fisheries Research
and Development Institute (2008) recently reported a possible new
species, referred to as Neodo-Daege, which often has a mixture of
characteristics of snow crabs and red snow crabs. Morphological
identification of the two species and the putative hybrid (Neodo-Daege)
has proved difficult, and has impeded the monitoring of wild harvests.
Despite the general prevalence of Chionoecetes species in Korea,
information about the occurrence of natural hybrids and native species
is scarce. The limited information currently available may not reflect
the true extent of hybridization, or may simply result from insufficient
attention having been paid to hybrid identification in the past. The
presence of hybrids can affect the TAC of both snow crabs and red snow
crabs, and can cause problems with the management of snow crab and red
snow crab wild resources. The regional management of crab resources
would therefore benefit from the development of effective species/hybrid
identification.

Hybridization between discrete species is a relatively common
natural phenomenon in wild populations of many species. Unfortunately,
no single morphological characteristic can be used to identify pure
species and hybrids. Hybrids often, however, display intermediate
morphological traits, although extreme or novel characteristics can also
be present in hybrid phenotypes (Karinen & Hoopes 1971). Although
morphological traits alone are often of limited value when identifying
natural hybrids, molecular markers have proved much more reliable
(Masaoka & Kobayashi 2005, Hurtado et al. 2011). Mitochondrial DNA
(mtDNA), because of its uniparental (maternal) mode of inheritance, is
not useful for the initial identification of Fl hybrids but, when
combined with nuclear markers, can allow the maternal parent of a hybrid
to be recognized and may provide evidence for inter-/intraspecific
hybridization.

The current study examined DNA sequence variation in the internal
transcribed spacer (ITS) region of nuclear ribosomal DNA and the mtDNA
cytochrome oxidase I (CO I) gene in populations of sympatric
Chionoecetes species and their putative hybrids to evaluate the origins
of hybrids. The study used morphological and molecular characteristics
to establish diagnostic markers specific to C. japonicus, C. opilio, and
their hybrids, sampled from a sibling complex in Korea.

MATERIALS AND METHODS

Sampling and DNA Isolation

A total of 94 samples (26 C. japonicus (23 males, 3 females), 26 C.
opilio (l 9 males, 7 females) and their putative hybrids (42; 34 males,
8 females), tentatively identified according to shell morphology, were
collected from the Ulzin coast of the East Sea in Korea. All individuals
were transferred to the laboratory on dry ice, and then muscle tissues
were soaked in TNES-urea buffer for DNA extraction. Total genomic DNA
was isolated from muscle tissue using the TNE--urea buffer method
(Asahida et al. 1996).

Morphological Diagnostic Characteristics

Three morphological traits were characterized for each individual
of each species: (1) the arrangement of granules on the lateral
carapace, (2) carapace color, and (3) the presence of spines on both
sides of the lateral margin of the carapace.

PCR Amplification and Sequencing

A partial mitochondrial CO I gene fragment was amplified using PCR
and universal primers (LCO: GGTCAACAAATCA TAAAGATATTGG and HCO:
AAACTTCAGGG-TGACC AAAAAATCA (Folmer et al. 1994)). Universal primers
(ITS5, 5'-GGAAGTA-AAAGTCGTAACAAGG-3' and ITS4, 5'-TCC
TCCGCTTATTGATATGC-3'), complementary to the conserved 18S and 28S
regions, respectively, were used to amplify the ITS region of the
nuclear rDNA gene, spanning ITS1, ITS2, and 5.8 rDNA (White et al.
1990). PCRs were performed in a 25-[micro]L volume containing 50 ng
genomic DNA, 10 mM Tris-HCl (pH, 8.0), 0.1% Triton X- 100, 50 mM KCl,
1.5 mM Mg[Cl.sub.2], 0.2 mM of each dNTP, 5 pmol of each primer, and 0.5
U Ex Taq DNA polymerase (Takara, Kyoto, Japan). PCR was performed in a
PTC-220 thermocycler (MJ Research, Watertown, MA) programmed for 5 min
at 95[degrees]C followed by 35 cycles of 30 sec at 94[degrees]C, 30 sec
at 55[degrees]C, and 30 sec at 72[degrees]C, with a final extension of
10 min at 72[degrees]C. Amplified products were purified using AMPure
beads (Agencourt Bioscience, Beverly, MA) according to the
manufacturer's protocol for sequencing. Purified products from 2
male individuals of putative hybrid status were cloned using the pGEM-T
Easy system (Promega, Madison, WI) according to the manufacturer's
protocol. From each of these, 5 clones were obtained, and plasmid DNA
was purified using Acroprep 96-well plates (Pall, East Hills, NY).
Direct sequencing of all individuals except for 2 putative hybrids was
conducted in both directions with the primers used for amplification.
Sequencing reactions were performed using the ABI BigDye Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) in a PTC-220
thermocycler under the following conditions: 40 cycles of 15 sec at
94[degrees]C, 20 sec at 48[degrees]C, and 4 min at 60[degrees]C. DNA
sequences were obtained with an ABI 3100x1 automated sequencer (Applied
Biosystems).

Sequence Alignment

Sequences were edited and aligned using LaserGene EditSeq software
(DNAStar, Madison, WI), and assembled with LaserGene SeqMan. Each
sequence was checked manually for accuracy. We aligned 1,506 bp and
1,582 bp of the nuclear ITS regions of C. japonicus (GenBank accession
no. HQ909101) and C. opilio (HQ909100), respectively, and 658 bp of the
mitochondrial CO I genes of C. japonicus (HQ909099) and C. opilio
(HQ909098). For each amplicon, forward and reverse sequences were
aligned to produce a consensus sequence. Multiple sequence alignments of
individual consensus sequences were constructed to detect single
nucleotide polymorphisms (SNPs).

PCR-Restriction Fragment Length Polymorphism

PCR products from the mitochondrial CO I gene were digested with 5
U Hinc II or Ssp I (New England Biolabs, Beverly, MA) at 37[degrees]C
for 12 h in a final volume of 25 [micro]L. Restriction fragments were
resolved using electrophoresis in 1.5% agarose gel in 1X TBE buffer. A
100-bp ladder was used as a molecular weight marker. After gels were
stained with ethidium bromide, fragments were visualized under a UV
transilluminator and photographed.

SNP Genotyping

SNP genotyping was conducted using TaqMan SNP genotyping assays
(Applied Biosystems). The PCR contained genomic DNA (20 ng), 1X TaqMan
universal PCR master mix, forward and reverse primers (900 [micro]mol/L
each), 200 nmol/L VIC-labeled probe, and 200 nmol/L FAM-labeled probe.
PCR primers and MGB TaqMan probes were designed using Primer Express
(Table 1). The probes were MGB probes designed specifically for TaqMan
allelic discrimination (Applied Biosysterns). PCR was performed in a
96-well plate with a reaction volume of 25 [micro]L using a PE 9700
thermal cycler (Applied Biosystems) programmed for 2 min at 50[degrees]C
and 10 min at 95[degrees]C, followed by 40 cycles of 30 sec at
95[degrees]C and 1 min at 60[degrees]C. Each 96well plate contained 94
samples of unknown genotype and 2 negative controls. Completed PCR
plates were read on a 7500 real-time PCR system (Applied Biosystems),
and analyzed using allelic discrimination sequence detection software
(Applied Biosystems). As confirmation of SNP genotyping, the ITS region
and CO I gene fragments from randomly selected samples were amplified by
PCR using the primers LCO, HCO, ITS4, and ITS5, and checked by direct
sequencing. Because the results of allelic discrimination were 100%
concordant with the direct sequencing, remaining genotyping was done
using the TaqMan system only.

RESULTS

The most obvious differences between C. japonicus and C. opilio
were carapace color, the arrangement of granules on the lateral
carapace, and the absence or presence of spines on the lateral carapace
(Table 2). Individuals that were intermediate for these morphologies
were designated as putative hybrids. The carapace color of C. japonicus
was vermilion, whereas that of C. opilio was dark brown, and the
putative hybrid was graybrown. The arrangement of granules on the
lateral carapace of C. japonicus included 2 parallel lines that merged
into a single line in the central region, whereas the parallel lines did
not converge in C. opilio. In putative hybrids, the 2 parallel lines
were closely aligned in the central region but not closed, suggesting
that they were intermediate between C. japonicus and C. opilio. Spines
on both sides of the lateral margins of the carapace were present in C.
japonicus and absent in C. opilio, but also present in the putative
hybrid. However, the discrimination of putative hybrids from either pure
C. japonicus or pure C. opilio using these key characteristics requires
careful observation.

The boundaries of the ITS region were determined by comparing them
with sequences from other species (FJ356675, FJ356678, FJ356682, and
HQ534061). Amplification of the whole ITS regions of C. japonicus and C.
opilio produced PCR products of 1,506 bp (32 bp 18S, 785 bp ITS1, 163 bp
5.8S, 487 bp ITS2, and 39 bp 28S) and 1,582 bp (32 bp 18S, 861 bp ITS1,
163 bp 5.8S, 487 bp ITS2, and 39 bp 28S), respectively. The length
differences between the PCR products of the 2 species resulted from a
76-bp deletion in ITS1 with 785 bp for C. japonicus, as shown in Figure
1. In ITS1, there were 87 polymorphic nucleotide sites (76 indels and 11
substitutions) between the 2 species (Fig. 1). For ITS 2, we aligned 487
bp and observed no indels, but there were 8 polymorphic nucleotide sites
(5 transversions and 3 transitions) between the 2 species (Fig. 2).
Polymorphic sites all distinguished C. japonicus haplotypes from those
of C. opilio. No intraspecific variation was evident for C. japonicus,
whereas C. opilio was polymorphic. All putative hybrids showed double
peaks at all 19 polymorphic sites for the ITS1 and ITS 2 sequences. Two
sequence types were found among the 10 ITS clones from putative male
hybrids. Six ITS clones matched sequences from C.japonicus, and 4
sequences were identical to those of C. opilio. Sequence alignments
showed that all hybrid individuals had the species-specific mutations
found in the two parental species. No novel ITS genotypes were detected
in hybrids, indicating that no species other than C. japonicus and C.
opilio were involved in the parentage of these hybrids.

A 658-bp partial sequence of the CO I gene was sequenced directly
in all sampled individuals. The CO I sequences obtained agreed with the
morphological identifications of the parental species: C. japonicus and
C. opilio. No indels were evident in any CO I sequence. Twenty-six
variable sites were found in the CO I sequences (Fig. 3), and these
sites all distinguished C. japonicus from C. opilio. No variation was
identified within the parental species. All putative hybrids had
sequences that were completely identical with C. japonicus. Two
restriction enzyme sites (Ssp I and Hinc II) were detected along the
sequence that could distinguish between C. japonicus and C. opilio.

For fast and accurate species identification and hybridization, we
examined one SNP located in the ITS1 region (334C in C. japonicus and
402A in C. opilio) and one SNP (58G in C. japonicus and 58A in C.
opilio) in the CO I gene in all 96 individuals (26 C. japonicus, 26 C.
opilio, and 42 putative hybrids) using the TaqMan SNP genotyping method.
The presence of SNPs was confirmed by direct sequencing of selected
samples. A scatter plot completely separated the two species (C.
japonicus and C. opilio) and their putative hybrids from each other
using an SNP assay of the ITS1 region (Fig. 4A). The SNP assay for the
CO I gene also showed complete separation of the two species, whereas
all putative hybrids grouped with C. japonicus (Fig. 4B). All putative
hybrids tested were heterozygous C/A representing both fixed alleles in
C. japonicus and C. opilio as parental species in the SNP genotyping of
ITS1, and were also homozygous G/G at the CO I gene, corresponding to
the C. japonicus genotype. The results confirmed that all putative
hybrids were indeed hybrids of C. japonicus and C. opilio, and suggest
that asymmetric hybridization was occurring.

The 708-bp PCR fragments of the CO I gene were digested with two
restriction enzymes: Ssp I and Hinc II. Restriction fragment length
polymorphism (RFLP) analysis showed two discrete banding patterns
corresponding with C. japonicus and C. opilio (Fig. 5). A substitution
from T to C at site 212 (based on the CO I sequence (Fig. 3)) caused a
loss of the Ssp I recognition site in C. opilio, and Ssp I cut the C.
japonicus PCR product once, generating bands of approximately 473 bp and
235 bp. A substitution from C to T at site 385 (based on the CO I
sequence (Fig. 3)) caused a loss of the Hint II recognition site in C.
opilio, whereas Hinc II cut the C. japonicus PCR product twice,
generating bands of approximately 4 bp, 301 bp, and 403 bp. A
restriction fragment of 4 bp could not be identified in agarose gel
analysis because of its small size. All 5 putative hybrids had the C.
japonicus RFLP pattern. This result confirmed that hybrids had sequences
identical to C. japonicus.

DISCUSSION

Hybrid identification based on morphology, ecology, and behavior
can be difficult, and thus both nuclear and mitochondrial molecular
markers have supplied valuable data for detecting hybridization events
as well as for identifying reciprocal hybridization (Imai & Takeda
2005, Gosling et al. 2008, Hashimoto et al. 2010). Although it is common
fisheries practice in Korea to produce interspecific hybrids for
culture, this is the first study that has used molecular markers to
verify natural hybridization events between C. japonicus and C. opilio
in the East Sea of Korea. PCR-RFLP (CO I) and SNP (ITS1) genotyping
strategies proved to be efficient methodologies, executed quickly and
inexpensively, that allowed for diagnosis via a simple PCR assay.

Both C. japonicus and C. opilio are distributed at operlapping
depth ranges in the East Sea. C. opilio also has a spawning season that
overlaps, at least in part, with that of C. japonicus (Lim et al. 2000,
Chun et al. 2008). Thus, because C. japonicus females carrying oocytes
have been found throughout the year, they might mate with C. opilio
males from March to April, which is the spawning season of snow crabs.
It would likely facilitate the potential for natural hybridization
between the two species in the East Sea. In our study, C. japonicus was
identified as the maternal species in hybrids because all partial CO I
sequences in the putative hybrids screened belonged to the C. japonicus
genotype. Because nuclear DNA is inherited from both parents, it is
possible to determine whether Neodo-Daege are hybrids of C. japonicus
and C. opilio using comparisons of their sequences. In general,
mitochondrial genes are normally inherited maternally, and are used to
identify the maternal ancestry of putative hybrids. The nuclear ITS data
clearly revealed polymorphic states in sequences obtained from direct
sequencing of the putative hybrids at each site where C. japonicus and
C. opilio showed fixed differences, supporting the idea of natural
hybridization between the two species.

Analysis of mitochondrial CO I haplotypes showed that all putative
hybrids had the C. japonicus haplotype, indicating that hybrids resulted
from unidirectional crosses between C. japonicus females and C. opilio
males. However, the possibility that some hybrids could result from
reverse crosses cannot be rejected. The reason why only C. japonicus
female x C. opilio male hybrids were detected in this study is not clear
to date. A plausible explanation is the possible lower survival and
fertility potential of C. opilio female x C. japonicus male hybrids.
Differences in the survival and fertility of reciprocal salmonid and
cyprinid F1 hybrids have been reported (Suzuki & Fukuda 1971, Mukai
et al. 2000). Furthermore, the presence of mature gametes in hybrids
also indicates that these individuals are potentially able to cross with
other hybrids and/or the parental species. The occurrence of both
species in the East Sea of Korea and the existence of their hybrids
suggest that this area may be a natural hybrid zone, but the collection
of more samples covering a broad range of natural habitat is required to
confirm this.

Hybridization between Chionoecetes bairdi and C. opilio has been
described previously, and F2 and later generations of hybrids were also
reported (Merkouris et al. 1998, Urban et al. 2002, Smith et al. 2005).
Previously, molecular markers have been used to resolve questions about
the hybridization and introgression of Chionoecetes species. The markers
used to confirm the origins of C. bairdi x C. opilio hybrids include
allozymes (Johnson 1976, Grant et al. 1978), the nuclear ITS1 region,
and the mtDNA 16S rRNA gene (Smith et al. 2005). Genetic markers have
also been used to investigate levels of genetic diversity in highly
exploited populations of Alaskan tanner crabs, C. bairdi, and Alaskan
and Atlantic snow crabs, C. opilio (Merkouris et al. 1998). The observed
concordance between the molecular and morphological data presented here,
in combination with findings by Urban et al. (2002) and Smith et al.
(2005), suggest that both techniques provide reliable identification of
red snow crabs, snow crabs, and their hybrids. Although the molecular
data strongly support the morphological data that intermediate
phenotypes are hybrids, a more detailed survey will be required of the
parental species and their hybrids in the East Sea of Korea with
additional markers that require the collection of more samples.

The SNP genotyping method has the advantage of allowing for
discrimination between genotype groups without requiting sequence
analysis. Its advantages include accuracy, ease of use, short duration,
and high-throughput analysis, but its disadvantage is that probe setting
is sometimes impossible. The current SNP genotyping method using Taqman
genotyping assays of the ITS1 and CO I gene regions allows for rapid and
accurate discrimination among genotype groups (C. japonicus, C. opilio,
and putative hybrids) without sequence analysis. The morphologically
intermediate samples found in the zone of sympatry all exhibited
heterozygote genotypes after SNP genotyping of the ITS1 region. Ongoing
experiments using the species-specific molecular markers developed
herein would help to quantify the degree of interspecific hybridization
and to determine the extent of gene introgression.

To be a useful management and conservation tool, the genetic
monitoring of hybridization must be applied in a routine manner. The
results of this study can serve as a framework for the characterization
of other crab hybrids and can contribute to studies of natural
populations, including the detection of natural hybridization events as
well as the verification of possible hybrid escapes into aquatic
environments from crab culture. Thus, we believe that the development
and examination of additional practical markers (nuclear genes) and the
collection of samples covering a broader geographical range will
increase the understanding of hybridization events between red snow and
snow crabs. Interspecific crosses between these species are being
performed currently in the laboratory to determine the relative
fertility and viability of reciprocal hybrids.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Fisheries
Research and Development Institute (RP-2011-BT-015).

(1) Biotechnology Research Division, National Fisheries Research
and Development Institute, Busan 619705, Republic of Korea; (2)
Fisheries Resources Management Division, National Fisheries Research and
Development Institute, Busan 619-705, Republic of Korea; (3) Dokdo
Fisheries Research Center, National Fisheries Research and Development
Institute, Pohang 791-119, Republic of Korea

* Corresponding author. E-mail: wjkim@nfrdi.go.kr

DOI: 10.2983/035.031.0106

TABLE 1.
TagMan PCR primers and probes used in genotyping of the internal
transcribed spacer 1 region and mitochondrial cytochrome oxidase
I gene.
Primer(5'-3')
Locus Forward Reverse Genotype Allele
ITS1 GCGCTGTTAGAGG GGGAGCCACTTGA C/A C
GTTTGTG ATGAACGAA A
COI GAGCTGGCATAGTT AGTTCCGGGTTGT G/A C *
GGTACATCATT CCAAGTTC T *
Locus Allelic Probe
ITS1 FAM-CTGTGCCTAGCTGCTGA-MGBNFQ
VIC-TCTGTGCCTAGATGCTGA-MGBNFQ
COI FAM-CTCGAATAATCAATCTT-MGBNFQ
VIC-AGCTCGAATAATTAATCTT-MGBNFQ
The positions of SNPs in the allelic probes are underlined.
* Probe sequences are those of the reverse strands.
TABLE 2.
Morphological differences of Chionoecetes japonicus, Chionoecetes
opilio, and putative hybrid.
Characteristics
Presence of Spine
Arrangement of at Both Sides of
Granules on Lateral Lateral Margin in Carapace
Species Carapace Carapace Color
C. japoniocus Two parallel lines Present Vermilion
are closed as one
line at the central
region
Putative hybrid Two parallel lines Present Gray-brown
are closely
narrowed at the
central region but
not closed
C. opilio Two parallel lines Absent Dark brown
are not closed
The circles indicate the spines of the lateral margin in carapace.
The ovals indicate the granules of the lateral margin in carapace.

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